Living cells require a stable rate of metabolism. Therefore in both cells and in the organisms containing the cells such regulation is under stringent control Approximately 50% of all energy in carbohydrates is converted into ATP and the rest is released as heat [Alberts et al., Molecular Biology of the Cell, (1989)]. The ATP, in turn, is used during growth for biosynthesis, or otherwise is used for work and the energy is released as heat. The release of this heat is used to warm the body in the form of temperature. Failure to maintain a stable metabolic level on an organismal level is a pathology referred to as fever, i.e., an increase in the temperature of an organism. Variations in metabolic levels are seen in a variety of pathological states including during bacterial or viral infections, in cancer, and in the general wasting away found during sepsis or cachexia.
The temperature of an organism is often taken as being indicative of the health of that organism. Even slight deviations in temperature may indicate a pathology. However the temperature of an individual cell cannot be assayed due to the limitation of current technology. Thus, it is not yet known to what extent cellular temperature varies with time within a cell or between two different cells. However, based on studies of large populations of cells there are good grounds to believe that some pathological states may be detectable by measuring temperature at the level of single cells.
Cellular metabolism may be regarded as the balance sheet for all of the enzymatic reactions occurring in the cell. Within this context, the net exothermic activity is seen as black body radiation, i.e., the heat that we assay when we measure the temperature of the cell. Prior studies of biological metabolic activity have used calorimetry which is applied at the level of an entire organism, or at the very least, an individual organ. Microcalorimetry can be applied to as few as 10 cells. However, there are severe limitations in the current technology limit resolving the heat production from single cells [Kemp, Thermal and Energetic Studies of Cellular Biological Systems, A. M. James, ed. (Bristol: Wright), pp. 147-166 (1987)]. The most sensitive technique applied to measuring metabolism in tumor cells is the Cartesian diver which can resolve hundreds of cells [Lutton and Kopac, Cancer Res., 31:1564-1569 (1971)]. This technique, however, is still too crude to resolve a heterogeneous population of cells, and it requires dissociation of the cells. Much of normal cell physiology is a dynamic process that requires cellular interaction in a three dimensional matrix. Many cellular activities are modified by cell-to-cell contact. All of this is lost when cells are dissociated. Recently, techniques have been developed to measure metabolites such as ATP, glucose and lactate in living cells [Hossman et al., Acta Neuropathologica, 69:139-147 (1986); Okada et al., Journal of Neurosurgery, 77:917-926 (1992)]. These techniques utilize photographic film [Hossman et al., 1986, supra; Okada et al., 1992, supra] or photon-counting cameras [Tamulevicius and Streffer, British Journal of Cancer, 72:1102-1112 (1995)] and have demonstrated considerable heterogeneity in metabolism in tumors when assayed with a millimeter spatial resolution.
Unfortunately, presently a number of important issues cannot be addressed because the metabolism of an individual cell cannot be determined. For example, we cannot presently study heterogenous populations of cells and resolve the activity of individual cells rather than the average of the mix. For example, a tumor is made of cancerous cells, normal cells, and infiltrating immune cells each of which is metabolizing at very different rates. In this case the measurement of the average metabolism of the tumor may not reflect the actual metabolism of the individual cells.
In most cells aerobic respiration is responsible for almost all of the production of ATP, with anaerobic glycolysis accounting for the remainder. It was noted over fifty years ago that anaerobic respiration is substantially increased in Ascites tumor cells relative to non-tumor cells [Warburg et al., The Metabolism of Tumors, ed. O. Warburg, Constable & Company LTD., London, pp. 129-170 (1930a); Warburg et al., The Metabolism of Tumors, ed. O. Warburg, Constable & Company LTD., London, pp. 254-265 (1930b)]. This led to the hypothesis that aerobic respiration was damaged in tumor cells [Warburg, Science, 123:309-314 (1956)]. As more has been learned about metabolism in the intervening years it was found that aerobic respiration is normal in tumor cells, but anaerobic respiration is increased. The mechanism responsible for this increase in anaerobic respiration is not presently understood. One major limiting factor in learning the mechanism is the inability to assay the relative metabolic levels of individual cells. As mentioned above, one particular problem is that tumors consist of a mix of normal, malignant, and immune cells. Current technology only allows the measurement of the average metabolism of this mixed population of cells. A second problem is the considerable heterogeneity even within the tumor cells. For example in tumors, oxygenation is often rate limiting for tumor growth [Kallinowski et al., J. Cel. Physiol., 138:183-191 (1989)]. Thus, in rapidly growing tumors growth is limited by angiogenesis, the growth of new blood vessels for the delivery of oxygen and nutrients.
Chemotherapy is a powerful tool that is used to fight tumors. However, tumor cells frequently develop resistance to the chemotherapeutics. This resistance is observed as a decreased sensitivity to a broad spectrum of chemotherapeutic agents and such cells have been labeled multi-drug resistant [Simon et al., Proc. Natl. Acad. Sci. USA, 91:3497-3504 (1994); Schindler et al., Biochemistry 35, 2811-2817 (1996); U.S. patent application No. 09/080,739, filed May 18, 1998; and U.S. Pat. No. 5,851,789, Issued Dec. 22, 1998, the contents of which are hereby incorporated by reference in their entireties]. Although these cells were originally viewed as "super cells" capable of withstanding any therapeutic challenge, there is now growing evidence that multi-drug resistant cells escape chemotherapy by behaving more like normal cells; and in some ways these cells appear to have undergone a "reverse" transformation in their properties [Biedler et al., Cancer & Metastasis Reviews, 13:191-207 (1994)]. Once chemotherapeutic drugs are removed, these cells resume their aggressive malignant properties. It is not known what happens to the metabolism of multi-drug resistant cells during this period. Indirect results indicate that the level of anaerobic respiration in these cells have returned to the levels seen in non-transformed cells [Miccadei et al., Oncology Research, 8:27-35 (1996)]. Therefore there is a need to measure the metabolism of individual cells that are multi-drug resistant in order to diagnose when tumors are shifting from their quiescent multi-drug resistant phase into a more malignant stage. Furthermore, there is a need to provide a method of measuring the temperature of individual cells to determine the metabolic changes that occur in either normal or tumor cells. In addition, there is a need of a methodology for measuring the temperature of a cell from a fresh biopsy of living tissue in order to rapidly diagnose the tissue for the presence of tumor cells.
The emission of almost all fluorophores is affected by temperature, however, the use of fluorophores that are particularly sensitive to temperature was first used as a technique for calibrating temperature by Kolodner et al., [Appl. Phys. Lett. 40:782-784 (1982); Appl. Phys. Lett. 42: 782-784, (1983)]. Kolodner used a fluorophore, Eu(TTA).sub.3 embedded in a polymer, PMMA, to achieve temperature resolutions of 0.07.degree. K. and of 10 .mu.M spatial resolution. This temperature-sensitive fluorophore thus could be used to quantify temperatures.
A variety of fluorophores, including Eu(TTA).sub.3 have been solubilized into various solvents and polymers and used to "paint" airplane wings in the laboratory of John P. Sullivan (Pursue University, see Table 1 modified from [Campbell et al., Temperature Sensitive Fluorescent Paint Systems, 18:94-2483 (1994)]). The change in fluorescence can be used to monitor the temperature changes of the airplane wing in a wind tunnel, for example. To date, temperature-sensitive fluorophores have been used in integrated circuit diagnostics [Kolodner et al., Appl. Phys. Lett., 42:117-119 (1983)], to detect boundary layer transition on a two-dimensional wing, and to visualize the interaction between the leading edge vortices and the surface of a delta wing [Campbell et al., in sixth Intern. Symp. on Applications of Laser Techniques to Fluid Mechanics, Lisbon, Portugal (1992); Campbell et al., Temperature Measurement Using Fluorescent Molecules, Abstract (1992); Campbell et al., Temperature Sensitive Fluorescent Paint Systems, 18:94 2483 (1994); Hamner et al., A Scanning Laser System for Temperature and Pressure Sensitive Paint, 32:94-0728 (1994)].
The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.